Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder causing cognitive, memory and behavioral impairments. It is the most common cause of dementia in the elderly population affecting roughly 5% of the population above 65 years and 20% above 80 years of age. AD is characterized by an insidious onset and progressive deterioration in multiple cognitive functions. The neuropathology involves both extracellular and intracellular argyrophillic proteineous deposits. The extracellular deposits, referred to as neuritic plaques, mainly consist in amyloid-beta (Aβ) peptides surrounded by dystrophic neurites (swollen, distorted neuronal processes). The Aβ peptides within these extracellular deposits are fibrillar in their character with a β-pleated sheet structure. Aβ in these deposits can be stained with certain dyes e.g. Congo Red and display a fibrillar ultrastructure. These characteristics, adopted by Aβ peptides in its fibrillar structure of neuritic plaques, are the definition of the generic term amyloid. Frequent neuritic plaques and neurofibrillary tangles deposits in the brain are diagnostic criteria for AD, as carried out when the patient has died. AD brains also display macroscopic brain atrophy, nerve cell loss, local inflammation (microgliosis and astrocytosis) and often congophilic amyloid angiopathy (CAA) in cerebral vessel walls.
Two forms of Aβ peptides, Aβ40 and Aβ42, are the dominant species of AD neuritic plaques (Masters et. al., 1985), while Aβ40 is the prominent species in cerebrovascular amyloid associated with AD (Glenner and Wong, 1984). Enzymatic activities allow these Aβ to be continuously formed from a larger protein called the amyloid precursor protein (APP) in both healthy and AD afflicted subjects in all cells of the body. Two major APP processing events β- and γ-secretase activities enables Aβ-peptide production through enzymatic cleavage, while a third one called α-secretase activities prevents Aβ-peptide by cleavage inside the Aβ-peptide sequence (reviewed in Selkoe, 1994; U.S. Pat. No. 5,604,102). The Aβ42 is forty two amino acid long peptide i.e. two amino acids longer at the C-terminus, as compared to Aβ40. The Aβ42 peptide is more hydrophobic, and does more easily aggregate into larger structures of Aβ peptides such as Aβ dimers, Aβ tetramers, Aβ oligomers, Aβ protofibrils or Aβ fibrils. Aβ fibrils are hydrophobic and insoluble, while the other structures are all less hydrophobic and soluble. All these higher molecular structures of Aβ peptides are individually defined based on their biophysical and structural appearance e.g. in electron microscopy, and their biochemical characteristics e.g. by analysis with size-exclusion chromatography/western blot. These Aβ peptides, particularly Aβ42, will gradually assemble into a various higher molecular structures of Aβ during the life span. AD, which is a strongly age-dependent disorder, will occur earlier in life if this assembly process occurs more rapidly in the brain of that individual. This is the core of the “amyloid cascade hypothesis” of AD which claims that APP processing, the Aβ42 levels and their assembly into higher molecular structures are central cause of all AD pathogenesis. All other neuropathology of AD brain and the symptoms of AD such as dementia are somehow caused by Aβ peptides or assembly forms thereof. The strongest evidence for the “amyloid cascade hypothesis” comes from genetic studies of individuals in families afflicted by early onset of familial AD as a dominant trait. These studies have revealed that rare mutations in the APP gene are sufficient to generate severe forms of AD. The mutations are clustered in and around Val 717 slightly downstream of the Aβ1-42 C-terminus (Goate et al., 1991, Chartier-Harlan, et al., 1991, Murrell, et al., 1991) and a unique double mutation (670-671) immediately upstream of the Aβ N-terminus in a Swedish family (Mullan, et al., 1992; U.S. Pat. No. 5,795,963). The APP mutations, which frames the Aβ peptide sequence, were later found to either increase both Aβ40 and Aβ42 production (the “Swedish” mutation; Citron, et al., 1992, Cai et al., 1993), or to increase the ratio of Aβ42/Aβ40 production and also to generate Aβ peptides that are C-terminally extended to incorporate the pathogenic mutation in the Aβ peptide e.g. Aβ50 (the “717”-mutations are at position 46; Suzuki et al., 1994; Roher et al., 2003). Thus the “717” mutations, in addition to wild-type Aβ40 and wild-type Aβ42, also generate London Aβ peptides (V717I) and Indiana Aβ peptides (V7171F, Aβ46 and longer forms of Aβ) which rapidly forms Aβ fibrils. In contrast, the Swedish mutation only generates increased levels of wild-type Aβ40 and Aβ42 peptides. Early onset familial AD is more often caused by mutations in presenilin 1 (on chromosome 14; U.S. Pat. No. 5,986,054; U.S. Pat. No. 5,840,540; U.S. Pat. No. 5,449,604) and in some cases by mutations in presenilin 2 (chromosome 1). Presenilin 1 and presenilin 2 are both polytopic transmembrane proteins that, together with three other proteins nicastrin, aph1 and pen-2, constitute the necessary functional core of the γ-secretase complex that enables Aβ-peptide formation through enzymatic cleavage of APP (Edbauer et al., 2003). All AD pathogenic mutations in presenilin 1 and presenilin 2 proteins significantly increase Aβ 1-42 overproduction (Schuener et al., 1996). Apolipoprotein E (ApoE) is, besides age, the most important risk factor for late-onset AD. There are three variants of the ApoE protein in humans, due to single amino acid substitutions in the ApoE protein. The ApoE4 variant confers increased risk of AD, while the ApoE2 variant is protective as compared to the predominant ApoE3 variant (Strittmatter et al., 1993; Corder et al., 1993). These protein changes are not deterministic, but confer enhanced or decreased susceptibility to develop AD in a population. The ability of the ApoE variants to facilitate amyloid deposition in APP transgenic mice models of AD is greatest for ApoE4, intermediate for ApoE3 and lowest for ApoE2, suggesting that the AD pathogenic mechanism of ApoE is to enhance Aβ-peptide assembly and/or amyloid deposition (Fagan et al., 2002). Other proteins such as α1-antichymotrypsin (Nilsson et al., 2001) and ApoJ/clusterin (DeMattos et al., 2002) also enhance Aβ-peptide assembly and/or amyloid deposition in APP transgenic mice, similar to ApoE. Neprilysin (NEP) and insulin-degrading enzyme (IDE) degrade Aβ peptides and are likely implicated in AD. However, none of these proteins has been proven to be involved in AD by human genetics. A key issue in future AD research is to better understand how enhanced levels Aβ or aggregates thereof cause dementia and functional loss in AD patients. It has been a long-standing belief that the insoluble amyloid fibrils, the main component of the neuritic plaque, are the pathogenic species in AD brain. High concentrations of Aβ fibrils have been shown to be cytotoxic in cell culture models of nerve cells in the brain (Pike et al., 1991; Lorenzo and Yankner et al., 1994). However, the hypothesis of the amyloid fibril as the main neurotoxic species is inconsistent with the poor correlation between neuritic plaque density and AD dementia score and also with the modest signs of neurodegeneration in current APP transgenic mice. Soluble neurotoxic Aβ-intermediate species and their appropriate subcellular site of formation and distribution could be the missing link that will better explain the amyloid hypothesis. This idea has gained support from recent discovery of the Arctic (E693) APP mutation, which causes early-onset AD (W00203911; Nilsberth et al., 2001). The mutation is located inside the Aβ peptide sequence. Mutation carriers will thereby generate variants of Aβ peptides e.g. Arctic Aβ40 and Arctic Aβ42. Both Arctic Aβ40 and Arctic Aβ42 will much more easily assemble into higher molecular structures of Aβ peptides that are soluble and not fibrillar in their structure, particularly Aβ protofibrils named LSAP (Large soluble amyloid protofibrils). Thus the pathogenic mechanism of the Arctic mutation differs from other APP, PS1 and PS2 mutations and suggests that the soluble higher molecular structures of Aβ peptides e.g. Aβ protofibrils is the cause of AD. It has recently been demonstrated that soluble oligomeric Aβ peptides such as Aβ protofibrils impair long-term potentiation (LTP), a measure of synaptic plasticity that is though to reflect memory formation in the hippocampus (Walsh et al., 2001). Furthermore that oligomeric Arctic Aβ peptides display much more profound inhibitory effect than wt Aβ on LTP in the brain, likely due to their strong propensity to form Aβ protofibrils (Klyubin et al., 2003).
An animal model of AD with the features of the human disease is much needed to better understand AD pathogenesis and to evaluate the efficacy of new therapeutic agents. The ideal animal model of AD should generate the complete neuropathology of AD and the clinical phenotype e.g. progressive memory and cognitive dysfunctions. Major progress in this direction has been accomplished using transgenic overexpression of APP harboring AD pathogenic mutations. Current APP transgenic models of AD display important features of AD pathogenesis such as age-dependent and region-specific formation of both diffuse and neuritic plaques in the brain. The amyloid pathology is associated with hyperphosphorylated tau, local inflammation (microgliosis and astrocytosis) and to a variable extent with congophilic amyloid angiopathy (CAA). These models have been generated by very high transgene expression of human APP, particularly in nerve cells of the brain. The transgenes always carries an AD pathogenic mutation. Thus a “717”-APP-mutation (V717F; Games et al. 1995; US2002104104; U.S. Pat. No. 5,720,936; U.S. Pat. No. 5,811,633) or the “Swedish” mutation (KM670/671NL; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997; WO 09803644; US2002049988; U.S. Pat. No. 6,245,964; U.S. Pat. No. 5,850,003; U.S. Pat. No. 5,877,399; U.S. Pat. No. 5,777,194) have been used. Double transgenic mice containing both mutant APP and mutant presenilin-1 transgenes develop accelerated amyloid plaques formation, but the animals still display modest mental impairment and still fail to display NFTs, nerve cell and brain atrophy (Holcomb et al., 1998; U.S. Pat. No. 5,898,094; US2003131364). Furthermore the current APP transgenic models likely have low levels of soluble intermediates in the Aβ fibrillization process such as Aβ protofibrils, which might be of great importance for AD pathogenesis. Several AD pathogenic mutations have previously been combined in one single transgene e.g. the “Swedish” mutation (KM670/671NL) and the “717”-APP-mutation (Indiana, V717F) have been used to enhance and increase formation of fibrillar Aβ peptides and neuritic plaque formation (Janus et al., 2001). Similarly the “Swedish” (KM670/671NL), the “Arctic” (E693G) and a “717”-APP-mutation (London, V717I) have been combined and used in an attempt to generate earlier and increased plaque formation (Teppner et al., 2003), like those of Swedish+Indiana APP transgenic models (Janus et al., 2001), since the London Aβ peptides will strongly facilitate Aβ fibril formation (Teppner et al., 2003; Roher et al., 2003). The unique characteristics of Arctic Aβ40 and Arctic A42 to form an abundance of stable protofibrils have been demonstrated (Nilsberth et al., 2001; Lashuel et al., 2003). The marked difference in pathology in human AD brain between carriers of the London APP mutation (Lantos et al., 1992; Cairns et al., 1993) and Arctic APP mutation reinforce the distinction in the chemical characteristics of London Aβ peptides and Arctic Aβ peptides for neuropathology.
The following references are presently found to be most relevant:    Stenh C. et al. disclose in “Metabolic consequences of the arctic (E693G) APP alzheimer mutation”, Society for Neuroscience. Abstract Viewer and Itenary Planner 2002, 32nd Annual Meeting of the Society for Neuroscience, Nov. 2-7, 2002, Abstract No. 296.6 and in Neuroreport 13, 1857-60 (2002) a transfected tumorigenic cell-line harboring APP cDNA with both the “Swedish” (KM670/671NL) and “Arctic” (E693G) mutations.    Hsiao et al., Science 274, 99-102 (1996) disclose a transgenic mouse harboring the “Swedish” (KM670/671NL) alone.    Mullan et al., Nature Genet. 1, 345-347 (1992) discloses the dominant inheritance of the “Swedish” (KM670/671NL) in a family with Alzheimer's disease.    Nilsberth et al., Nat. Neurosci. 4, 887-893 (2001) discloses the dominant inheritance of the “Arctic” (E693G) in a family with Alzheimer's disease.    Teppner et al., 6th Internat. Conf. AD/PD, Seville, Spain, board no 52 (2003), discloses a preliminary attempt to generate a transgenic mouse harboring the “Swedish” (KM670/671NL), “Arctic” (E693G) and “London” (V717I) mutations. No pathology is described.    Roher et al., J Biol Chem. 279(7): 5829-36 (2004), discloses that Aβ peptides extend beyond amino acid 42, e.g. Aβ 1-46 and Aβ1-50, in Alzheimer brain tissue from patient carrying a “London”-type mutation (V717F).    Kang et al., Nature 325, 733-6 (1987) describes the cloning of human APP695 cDNA.